Genotyping single point mutations in rd1 and rd8 mice using melting curve analysis of qPCR fragments.

PCR is tolerant to single nucleotide mismatches. Therefore, genotyping of point mutations by PCR requires special conditions for the amplification of allele-specific PCR fragments. MS-PCR (mutagenically separated PCR) is an improved version of ARMS (amplification refractory mutation system) in which additional nucleotide mismatches near the mutation site are used to separate the wt fragments from the mutant fragments in a single-tube PCR. In the originally described procedure, the resulting fragments are resolved on agarose gels according to differences in size introduced by different lengths of the allele-specific primers. In order to evaluate the PCR fragments by melting curve analysis, we enlarged the difference in the melting temperatures of the fragments of the two alleles by increasing the GC content of the longer allele-specific primer resulting in a higher melting temperature of the corresponding fragment. Using the murine retinal degeneration mutations rd1 and rd8 as an example, we show that such primers result in an easy to handle genotyping procedure: qPCR followed by melting curve analysis. In summary, MS-PCR is a simple and easy-to-use method for detecting single nucleotide variants.

Genetic variants related to insulin metabolism are associated with gestational diabetes mellitus.

The current study sought to investigate the associations of common genetic risk variants with gestational diabetes mellitus (GDM) risk in the north Indian population and to evaluate their utility in identifying GDM cases. A case-control study, including 300 pregnant women, was included, and clinical and pathological information was collected. The amplification-refractory mutation system (ARMS) was used for genotyping four single nucleotide polymorphisms (SNPs), namely FTO (rs9939609), PPARG2 (rs1801282), SLC30A8 (rs13266634), and TCF7L2 (rs12255372). The odds ratio and confidence interval were determined for each SNP in different genetic models. Further, attributable risk, population penetrance, and relative risk were also calculated. The risk allele A of FTO (rs9939609) poses a two times higher risk of GDM (p = 0.02, OR = 2.5). The CG and GG genotypes of PPARG2 (rs1801282) have half a lower risk of GDM. In SLC30A8 (rs13266634), the recessive model analysis showed a two times higher risk of having GDM, while the recessive model (TT vs. GG + GT) analysis in TCF7L2 (rs12255372) indicates a lower risk of GDM. Finally, the relative risk, population penetrance, and attributable risk for risk allele in all four variants was higher in GDM mothers. All four polymorphisms were found to be significantly associated with BMI, HbA1c, and insulin. Our study first time confirmed a significant association with GDM for four variants, FTO, PPARG2, SLC30A8, and TCF7L2, in the North Indian population.

Where do obesity and male infertility collide?

The parallel rise in obesity and male infertility in modern societies necessitates the identification of susceptibility genes underlying these interconnected health issues. In our study, we conducted a comprehensive search in the OMIM database to identify genes commonly associated with male infertility and obesity. Subsequently, we performed an insilico analysis using the REVEL algorithm to detect pathogenic single nucleotide polymorphisms (SNPs) in the coding region of these candidate genes. To validate our findings in vivo, we conducted a comprehensive analysis of SNPs and gene expression of candidate genes in 200 obese infertile subjects and 240 obese fertile individuals using ARMS-PCR. Additionally, we analyzed 20 fertile and 22 infertile obese individuals using Realtime-qPCR. By removing duplicated queries, we obtained 197 obesity-related genes and 102 male infertility-related genes from the OMIM database. Interestingly, the APOB gene was found in common between the two datasets. REVEL identified the rs13306194 variant as potentially pathogenic with a calculated score of 0.524. The study identified a significant association between the AA (P value = 0.001) genotype and A allele (P value = 0.003) of the APOB rs13306194 variant and infertility in obese men. APOB expression levels were significantly lower in obese infertile men compared to obese fertile controls (p < 0.01). Moreover, the AA genotype of rs13306194 APOB was associated with a significant decrease in APOB gene expression in obese infertile men (p = 0.05). There is a significant association between the Waist-to-Hip Ratio (WHR) and LH with infertility in the obese infertile group. These results are likely to contribute to a better understanding of the causes of male infertility and its association with obesity.

Multiplex fluorescent amplification-refractory mutation system PCR method for the detection of 10 genetic defects in Holstein cattle and its comparison with the KASP genotyping assay.

The common deleterious genetic defects in Holstein cattle include haplotypes 1-6 (HH1-HH6), haplotypes for cholesterol deficiency (HCD), bovine leukocyte adhesion deficiency (BLAD), complex vertebral malformation (CVM) and brachyspina syndrome (BS). Recessive inheritance patterns of these genetic defects permit the carriers to function normally, but homozygous recessive genotypes cause embryo loss or neonatal death. Therefore, rapid detection of the carriers is essential to manage these genetic defects. This study was conducted to develop a single-tube multiplex fluorescent amplification-refractory mutation system (mf-ARMS) PCR method for efficient genotyping of these 10 genetic defects and to compare its efficiency with the kompetitive allele specific PCR (KASP) genotyping assay. The mf-ARMS PCR method introduced 10 sets of tri-primers optimized with additional mismatches in the 3' end of wild and mutant-specific primers, size differentiation between wild and mutant-specific primers, fluorescent labeling of universal primers, adjustment of annealing temperatures and optimization of primer concentrations. The genotyping of 484 Holstein cows resulted in 16.12% carriers with at least one genetic defect, while no homozygous recessive genotype was detected. This study found carrier frequencies ranging from 0.0% (HH6) to 3.72% (HH3) for individual defects. The mf-ARMS PCR method demonstrated improved detection, time and cost efficiency compared with the KASP method for these defects. Therefore, the application of mf-ARMS PCR for genotyping Holstein cattle is anticipated to decrease the frequency of lethal alleles and limit the transmission of these genetic defects.

Determination of IL-23 receptor expression and gene polymorphism (rs1884444) in Iranian patients with ankylosing spondylitis.

BACKGROUND: Through investigating genetic variations, it has been demonstrated that single nucleotide polymorphisms (SNPs) in the IL-23 receptor (IL23R) gene have a critical role in the pathophysiology of ankylosing spondylitis (AS). Here, we investigated whether the IL23R variant (rs1884444) is associated with AS in the Iranian population. METHODS AND MATERIAL: In this research, we analyzed rs1884444 in a group of 425 patients with AS and 400 matched controls. For DNA extraction, the phenol/chloroform technique was utilized. Peripheral blood mononuclear cells (PBMCs) were obtained from the whole blood of 39 patients and 43 healthy controls and total RNA was extracted. Genotyping was performed by amplification-refractory mutation system (ARMS)-PCR method. Afterward, the expression level of IL23R was analyzed by the real-time quantitative (Q)-PCR method. RESULTS: We observed no significant association between the distribution of alleles and genotypes of rs1884444 and susceptibility to AS. In addition, the expression level of IL23R did not differ between PBMCs from AS patients compared to the control group (P = 0.167). Furthermore, the relative expression level of IL23R was positively correlated with the BASDAI (P < 0.01) and BASFI (P < 0.05) scores of the patients. CONCLUSION: It appears that IL23R polymorphism (rs1884444) and the level of gene expression might not contribute to the susceptibility to AS in the Iranian population. The correlation of IL23R expression with the level of BASDAI and BASFI scores in patients may be due to the role of the IL-23/IL-23R signaling cascade in inflammation and exert a critical role in the development of AS.

Spectrum of Rare and Novel Indel Mutations Responsible for ß Thalassemia in Eastern India.

There is limited data available regarding the clinical utility of routine molecular diagnosis of ß Thalassaemia in addition to HPLC-based screening in low resource settings. The current study highlights the caveats of an HPLC-based screening compared to the inclusion of genetic confirmation as a second-tier test and its implications in terms of genotype-phenotype correlation. A prospective, institution-based, observational study was conducted at the Department of Paediatric Medicine, including 103 children aged up to 12 years. Five common mutations for ß Thalassemia and the HbE mutation in the HBB gene were tested by a two-tiered approach using multiplex ARMS PCR and PCR RFLP methods respectively. Sanger sequencing of all three exons of the HBB gene was performed in all negative cases. Sequencing revealed many rare pathogenic mutations like c.316-106 C > G (dbSNP: 34,690,599); Hb Kairouan (c.92G > C); c.33 C > A (dbSNP rs35799536); c.47G > A (dbSNP rs63750783); c.51delC (HbVar ID 799); c.[93-2 A > C] and c.118 C > T (HbVar ID 845). We detected a novel Pathogenic M_000518.5(HBB):c.164_168delinsGGCATCA (p.Val55fs) mutation in a heterozygous state which was reported in the ClinVar database with accession ID VCV000590977.2. We also encountered several cases of silent carrier on HPLC and de novo occurrence of mutation. We conclude that the multiplex touchdown ARMS PCR methodology employed in the present study provides a low-cost solution for molecular diagnostics of Β Thalassaemia. The problem of silent carriers in HPLC is significant enough to rethink if we need supplemental genetic testing in the couple when one of the partners is a carrier. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01098-w.

Factor V Leiden, MTHFR, and FXIIIVal34Leu gene polymorphisms and their association with clinical features and risk of diabetic retinopathy in patients with type 2 diabetes.

Background: Diabetic retinopathy (DR) is expanding to epidemic levels globally due to the progressing prevalence of diabetes mellitus (DM). In this study, the association between factor V Leiden (FVL), MTHFRC677T, and FXIIIVal34Leu polymorphisms and diabetic retinopathy was investigated in Eastern Iran. Methods: This case-control study enlisted the participation of 300 people (diabetic patients=100, diabetic retinopathy patients=100, healthy controls=100), and polymorphisms were examined by Tetra primer ARMS-PCR. Results: The frequency of FVL (p=0.294) and FXIIIVal34Leu (P=0.349) polymorphism showed no significant results between the genotype frequency in the mentioned groups. In contrast, MTHFRC677T SNP was significantly different in diabetic patients and controls (P=0.008). The MTHFRC677T polymorphism was found to be connected with increased systolic blood pressure in patients who had the TT genotype (130.96±11.92mm/Hg; P=0.011). Conclusion: Our study recommended that the MTHFRC677T polymorphism may offer to DR development. Studies with larger sample sizes and a wider spectrum of populations are authorized to verify this finding.

The Association of miRNA-146a Gene Variation and Multiple Sclerosis in The Iranian Population.

Background: Multiple sclerosis (MS) is a complex human autoimmune-type inflammatory disease of the central nervous system (CNS). MicroRNA-146a (miR-146a) belongs to an endogenous and non-coding RNA family with 18-22 nucleotides long, which modulates the innate and adaptive immune response. Methods: Our study aimed to investigate a possible association between rs2910164 and rs2431697 polymorphisms of the miR-146a gene and multiple sclerosis in the Iranian population. A total of 60 MS cases and 100 controls were recruited. Single nucleotide polymorphism (SNP) rs2431697 was genotyped by utilizing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and SNP rs2910164 was genotyped by using Tetra-primer ARMS-PCR. Statistical Analysis conducted by the chi-squared test utilizing SPSS version 21.0 Software. The Hardy-Weinberg equilibrium assumption was evaluated. Results: The results of the present study suggest the miR-146a gene rs2431697 polymorphism is not associated with multiple sclerosis. However, there is a significant relationship between polymorphism rs2910164 of the miR-146a gene and multiple sclerosis in the population studied (P = 0.012). Conclusion: Our data provide evidence that the miR-146a gene may be involved in creating the susceptibility to MS in the Iranian population.

Targeted next-generation sequencing of 491 lung cancers in clinical practice: Implications for future detection strategy and targeted therapy.

Although lung cancer remains the most common cause of global cancer-related mortality, the identification of oncogenic driver alterations and the development of targeted drugs has dramatically altered the therapeutic landscape. In this retrospective study, we found that 97.7% samples carried at least one mutation in the 25 genes tested in our cohort. 53.6% samples were positive for EGFR mutations, followed by TP53 (41.1%), KRAS (11.8%), ERBB2 (4.3%). EGFR mutations were mainly found in female adenocarcinomas, while TP53 was mainly found in male non-adenocarcinomas. Significant differences can be found in the mutation rate of EGFR (60.9% vs 11.9%), KRAS (12.2% vs 25.0%), STK11 (1.5% vs 11.9%), FGFR3 (2.4% vs 0.0%) and ERBB4 (1.2% vs 6.1%) between adenocarcinoma in our cohort and TCGA-LUAD data (all p < 0.001). What's more, we found that the mutation of EGFR increased significantly from adenocarcinomas in situ (AIS, 21.4%) to microinvasive adenocarcinomas (MIA, 52.4%) and invasive adenocarcinomas (IA, 61.1%), while the mutation of ERBB2 dropped markedly from AIS (21.4%) to MIA (9.5%) and IA (4.1%). At last, comparations between targeted NGS and ARMS-based single gene test in the detection of EGFR showed a 94.6% consistence. In conclusion, targeted NGS can provide a comprehensive mutational profile of lung cancer. Considering the high mutation rate of EGFR in NSCLC of Asian populations, a specialized detection strategy should be conducted.

Micronutrients intake and genetic variants associated with premature ovarian insufficiency; MASHAD cohort study.

BACKGROUND AND AIM: premature ovarian insufficiency (POI) is defined as the menopause before 40 years of age, and its prevalence is reported to be two-fold higher in Iranian women than the average for woman globally. POI is associated with several cardio/cerebrovascular complications as well as an increased overall mortality. Genetic factors, and serum levels of minerals and vitamin D, have been reported to be related to the prevalence of POI. We have investigated the association between some POI -related genotypes with the serum levels of some important micronutrients. METHODS: One hundred and seventeen women with POI and 183 controls without any renal, hepatic, and thyroid abnormalities were recruited as part of the MASHAD study. Demographic and anthropometric features were recorded and blood samples were collected and processed. DNA was extracted from the buffy coat of blood samples from all participants and 8 POI-related single nucleotide polymorphisms (SNPs) were determined using ASO-PCR or Tetra ARMS-PCR. Serum minerals and vitamin D concentrations were measured using routine methods. RESULTS: In women with POI, serum copper, phosphate, and calcium were significantly different for those with rs244715, rs16991615, and rs4806660 genotypes, respectively. In our control population, significant differences were also found in serum copper concentrations between different genotypes of rs4806660, rs7246479, rs1046089, and rs2303369. After adjusting for all confounding factors, the women with POI carrying TC genotype (rs4806660) had a lower risk to have serum copper levels < 80 (µg/dL) than those carrying a TT genotype. Furthermore, women with POI carrying GG genotype (rs244715) had a 6-fold higher risk to have serum copper levels > 155 than those carrying AA genotype. CONCLUSION: The C and G alleles of the rs4806660 and rs244715 polymorphisms respectively are independently associated with serum copper in women with POI. Further studies are necessary to investigate the association of serum copper and other micronutrients in women and other POI -related polymorphisms.

Integrated approach for detection of SARS-CoV-2 and its variant by utilizing LAMP and ARMS-PCR.

Global impact of COVID-19 pandemic has heightened the urgency for efficient virus detection and identification of variants such as the Q57H mutation. Early and efficient detection of SARS-CoV-2 among densely populated developing countries is paramount objective. Although RT-PCR assays offer accuracy, however, dependence on expansive kits and availability of allied health resources pose an immense challenge for developing countries. In the current study, RT-LAMP based detection of SARS-Cov-2 with subsequent confirmation of Q57H variant through ARMS-PCR was performed. Among the 212 collected samples, 134 yielded positive results, while 78 tested negative using RT-LAMP. Oropharyngeal swabs of suspected individuals were collected and processed for viral RNA isolation. Isolated viral RNA was processed further by using either commercially available WarmStart Master Mix or our in house developed LAMP master mix separately. Subsequently, the end results of each specimen were evaluated by colorimetry. For LAMP assays, primers targeting three genes (ORF1ab, N and S) were designed using PrimerExplorer software. Interestingly, pooling of these three genes in single reaction tube increased sensitivity (95.5%) and specificity (93.5%) of LAMP assay. SARS-CoV-2 positive specimens were screened further for Q57H mutation using ARMS-PCR. Based on amplicon size variation, later confirmed by sequencing, our data showed 18.5% samples positive for Q57H mutation. Hence, these findings strongly advocate use of RT-LAMP-based assay for SARS-CoV-2 screening within suspected general population. Furthermore, ARMS-PCR also provides an efficient mean to detect prevalent mutations against SARS-Cov-2.

Rapid Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction Protocol for Detection of Y132F Mutation in Fluconazole Resistant Candida parapsilosis.

There is an emerging fluconazole resistance in Candida parapsilosis in recent years. The leading mechanism causing azole resistance in C. parapsilosis is the Y132F codon alteration in the ERG11 gene which encodes the target enzyme of azole drugs. In this study, we evaluated the sensitivity, compatibility, and specificity of a novel tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method for rapid detection of the Y132F mutation in fluconazole nonsusceptible C. parapsilosis. Antifungal susceptibility tests for detection of fluconazole resistance were performed by broth microdilution according to the CLSI guidelines. All susceptible and nonsusceptible C. parapsilosis isolates were analyzed for ERG11 mutations with Sanger sequencing. T-ARMS-PCR was fully concordant with the Sanger sequencing (100% of sensitivity and specificity) for detection of Y132F mutations. T-ARMS-PCR method could be a rapid, simple, accurate, and economical assay in the early detection of the most common cause of fluconazole resistance in C. parapsilosis isolates. In routine laboratories with high C. parapsilosis isolation rates, performing the T-ARMS-PCR for early detection of the most common reason of fluconazole resistance in C. parapsilosis, could be a life-saving approach for directing antifungal therapy before obtaining the definitive antifungal susceptibility tests results.

The linkage between IL-6 rs1800797 variant and breast cancer susceptibility in Bangladeshi women: A case-control study.

Background and Aims: Breast cancer is one of the deadliest diseases affecting women in Bangladesh, and its prevalence is increasing year by year. Although several IL-6 single nucleotide polymorphisms have been implicated in BC susceptibility and prognosis in various studies, no research has been done to investigate the relationship between breast cancer and IL-6 in Bangladeshi women. This investigation aimed to explore the linkage between the rs1800797 variant of IL-6 and the susceptibility to breast carcinoma among women in Bangladesh. Methods: The IL-6 rs1800797 variant was genotyped in 218 subjects (110 cases and 108 controls) using the tetra-primer ARMS-PCR method. The statistical analysis was applied utilizing the SPSS software version 24.0. UALCAN database was used for IL-6 mRNA analysis, and genotype-based gene expression was retrieved from GTEx Portal. Results: This study found a significant link between IL-6 rs1800797 variants and increased chance of breast cancer across different genetic inheritance models, including additive model 1 (AG vs. GG: OR = 2.16, p = 0.035); dominant model (AG + AA vs. GG: OR = 2.26, p < 0.05); overdominant model (AG vs. GG + AA: OR = 2.08, p < 0.05); and allelic model (A vs. G: OR = 2.15, p < 0.05). However, an insignificant association of breast cancer was found in both additive model 2 (AA vs. GG: OR = 2.91, p > 0.05) and the recessive model (AA vs. GG + AG: OR = 2.52, p > 0.05). Under the analysis of the probability of false positive reports, no significant values were found in different models when the OR was 1.5, and the prior probability was 0.25. Conclusions: A significant relationship was found between the IL-6 rs1800797 genetic variant and the risk of breast cancer. However, the findings of the study should be further investigated with a larger sample size to validate the correlation.

Investigating the impact of the genetic variant CXCR1 (rs2234671) in individuals with urinary tract infections.

BACKGROUND: Urinary tract infections (UTIs) are currently posing a worldwide health concern by affecting millions of people. The genetic variant rs2234671 in the CXCR1-interleukin-8 receptor is closely related to a raised UTI risk. OBJECTIVES: In this work, the impact of CXCR1 (rs2234671) on UTI individuals was examined. METHODS: The demographic features of 30 recurrent UTI patients and 20 controls were thoroughly investigated. Bacterial isolation and identification were performed by the implementation of cultural and biochemical methods. DNA extraction, purification of all samples from both patients and healthy people, and IL-8 rs2234671 (C/G) SNP genotyping using T-ARMS-PCR were performed. The significance of the results was evaluated by carrying out a statistical analysis. FINDINGS: The patient's average age was 34.63 ± 11.44 years, and controls averaged 30.30 ± 8.59 years (P= 0.156). No significant gender difference existed (P= 0.804). Escherichia coli (63.3%) was predominant, followed by Proteus mirabilis (26.7%), Enterococcus faecalis (23.3%), Klebsiella pneumoniae (10.0%), and Pseudomonas aeruginosa (20.0%). No significant association was found between bacterial species frequency, age, or sex. From the CXCR1 (rs2234671) frequency comparison, a higher GG genotype incidence in UTI patients than controls was extracted (26.7% vs. 15.0%), though not statistically significant. Risk analysis revealed that GG homozygous and C/G heterozygous genotypes were not UTI risk factors (OR = 2.47 and OR = 1.85, respectively). Moreover, the allele frequencies displayed no significant difference between the patients and controls (G allele: 66.7% vs. 66.7%; C allele: 33.3% vs. 33.3%). MAIN CONCLUSIONS: Although no significant association between CXCR1 (rs2234671) and UTI was found, the GG genotype may point to the increasing probability of UTI risk. Additional research is required to confirm and expand these conclusions.

Genetic variation of CYP2C9 gene and its correlation with cardiovascular disease risk factors.

BACKGROUND: The major enzyme that is responsible for Sulfonylureas (SUs) metabolism is hepatic cytochrome P-450 2C9 (CYP2C9). It is encoded by the polymorphic gene CYP2C9, which has many allelic variants, among those the CYP2C9*2 and CYP2C9*3 are the most common and clinically significant allelic variations. People with diabetes mellitus type 2 (T2DM) are more likely to develop cardiovascular disease (CVD), and their risk of dying from it is more than two times higher than that of people without the condition. The purpose of this study was to evaluate the association of genetic variations in the CYP2C9 gene with cardiovascular risk factors by investigating CYP2C9*1, *2, *3, *5, *11, and *13 allelic variants. METHODS AND RESULTS: A total of 226 participants were enrolled in the current case-control study. Allele-specific amplification- PCR (ASA-PCR) was used to determine the allele of different variations and the results were confirmed by sequencing. The findings of this study showed the presence of the CYP2C9*2 allele in the T2DM group does not differ from its percentage in the control group. Also, CYP2C9*3 allele frequencies identified by Hardy-Weinberg equilibrium (HWE) analysis law were not significant, p = 0.6593 and 0.5828 in T2DM and control groups. There is no statistically significant difference between the control and diabetes groups involving the distribution of CYP2C9 alleles and CYP2C9*5, *11, and *13 polymorphisms were absent in the Iraqi population. No carrier for the CYP2C9*3 homozygous state was found in both groups. CONCLUSIONS: According to these results T2DM patients with the CYP2C9*2 and *3 variants have an increased risk of developing hypertension.

The miRNA variants MIR196A2 (rs11614913) and MIR423 (rs6505162) contribute to an increase in the risk of myocardial infarction.

INTRODUCTION: MicroRNAs (miRNAs) are small, single-stranded RNA molecules that negatively regulate gene expression and play a key role in the pathogenesis of human diseases. Recent studies have suggested that miRNAs contribute to cardiovascular diseases (CVDs). However, the association between single-nucleotide polymorphisms (SNPs) in miRNAs and myocardial infarction (MI) remains in infancy. AIM: The current study was designed to find out the association of SNPs in MIR196A2 and MIR423 (rs11614913 and rs6505162, respectively). METHODS: Using Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction (T-ARMS PCR) in 400 cases (MI patients) and 336 healthy controls. Using different inheritance models (co-dominant, homozygous dominant, homozygous recessive, and additive models), the association of these SNPs was genotyped with MI risk. RESULTS: For variant rs11614913, significant distribution of the genotypes among the cases and controls was determined by co-dominant [χ2 = 29.19, 2; p value < 0.0001], dominant (C/C vs. C/T + T/T) [OR = 0.45 (0.34 to 0.61); p < 0.0001], recessive (T/T vs. C/T + C/C) [OR = 1.009 (0.63 to 1.63); p-value p value > 0.999], and additive models [OR = 0.65 (0.52 to 0.80); p value = 0.0001]. Similarly, a significant association of rs6505162 was determined by co-dominant [χ2 = 24.29, 2; p value < 0.0001], dominant (C/C vs. A/C+ A/A) [OR = 0.44 (0.32 to 0.61); p value < 0.0001], recessive (A/A vs. A/C + C/C) [OR = 1.29 (0.85 to 1.98); p value = 0.28], and additive models [OR = 0.65 (0.52 to 0.81); p value = 0.0001]. CONCLUSION: Therefore, the current study showed that both variants rs11614913 and rs6505162 are significantly associated with MI in the Pakistani population.

Impact of IL-6 and IL-1ß Gene Variants on Non-small-cell Lung Cancer Risk in Egyptian Patients.

Lung cancer is a serious health and life issue, with the fastest-growing incidence and fatality rates worldwide. It is now clear that inflammation is a key factor involved in all aspects of carcinogenesis, notably lung cancer development. Genetic changes, including polymorphisms in inflammatory genes, are supposed to be a significant cause of increased lung cancer risk. The main idea of this research was to disclose the linkage between both IL-6 rs1800795 and IL-1ß rs16944 variants and susceptibility to non-small-cell lung cancer (NSCLC) in Egyptians. This case-control design was composed of 127 cases and 138 controls, which were genotyped using the ARMS-PCR technique. To examine the NSCLC susceptibility under various genetic models, the odds ratio (OR) and 95% confidence intervals (CIs) were determined by logistic regression. Rs1800795 of the IL-6 gene was linked to higher odds of NSCLC under the allele model (adjusted, OR 2.28; 95% CI 1.2-4.33; p = 0.011). In the genetic models, IL-6 rs1800795 elevated the odds of NSCLC, while IL-1ß rs16944 decreased the odds of NSCLC. Stratification analysis showed that IL-6 rs1800795 greatly increased the NSCLC risk in females and adenocarcinoma subtypes, whereas IL-1ß rs16944 largely decreased the NSCLC risk for males, patients aged < 55, and nonsmokers. Regarding clinical data, the IL-6 variant was remarkably correlated with tumor size. This work primarily established that IL-6 and IL-1ß variants have a great impact on NSCLC development in the Egyptian population; thus, it may be a supportive guide for earlier NSCLC prevention.

Deiodinase Type 3 Polymorphism (rs1190716) Affects Therapeutic Response to Levothyroxine.

Objectives: Levothyroxine (LT4) is a commonly used treatment for hypothyroidism. Deiodinase enzymes control the metabolism and homeostasis of thyroid hormones (THs). Deiodinase type 3 gene (DIO3) encodes deiodinase type 3 enzyme (D3), and the genetic polymorphisms of this gene could affect the levels of THs and the response to LT4 treatment. This study aimed to investigate the single-nucleotide polymorphism (SNP), rs1190716; C > T, of DIO3 as a candidate genetic variant that might affect the clinical response to LT4 treatment. Materials and Methods: Two hundred Iraqi hypothyroid female patients aged 40 years were enrolled in this cross-sectional study. All of them were already on the LT4 treatment for at least 4 months. THs [thyroxin (T4), triiodothyronine (T3), reverse triiodothyronine (rT3), and diiodothyronine (T2)] were estimated. An allele-specific polymerase chain reaction technique was performed to detect the rs1190716; C > T SNP. Results: The genotypes distribution of rs1190716; C > T SNP was 10 (4.5%) for the wild type (CC), 50 (22.7%) for the heterozygous mutant type (TC), and 160 (72.7%) for the homozygous mutant type (TT). The patients were divided into three groups according to their genotypes. Significant differences were found in the T4, T3, and T2 levels among the patients (p=0.019, p=0.039, p=0.032, respectively). Conclusion: The rs1190716; C > T SNP could affect the activity of the D3 enzyme and the metabolic homeostasis of the THs; therefore rs1190716; C > T SNP could have an impact on the therapeutic response to LT4 in Iraqi female patients with primary hypothyroidism. Regarding DIO3 gene, this is a novel finding; hence, further studies are needed to confirm it.

Differential Expression of Serum Proinflammatory Cytokine TNF-α and Genetic Determinants of TNF-α, CYP2C19*17, miR-423 Genes and Their Effect on Coronary Artery Disease Predisposition and Progression.

Coronary artery disease (CAD) is the leading cause of death and hospitalization worldwide and represents a problem for public health systems everywhere. In Saudi Arabia, the prevalence of CAD is estimated to be 5.5%. Risk factors for CAD include older age, male gender, obesity, high blood pressure, smoking, diabetes, hyperlipidemia, and genetic factors. Reducing the risk factors in susceptible individuals will decrease the prevalence of CAD. Genome wide association studies have helped to reveal the association of many loci with diseases like CAD. In this study, we examined the link between single nucleotide variations (SNVs) of TNF-α-rs1800629 G>A, CYP2C19*17 (rs12248560) C>T, and miR-423 rs6505162 C>A and the expression of TNF-α with CAD. We used the mutation specific PCR, ARMS-PCR, and ELISA. The results showed that the A allele of the TNF-α rs1800629 G>A SNP is linked to CAD with odd ratio (OR) (95% CI) = 2.10, p-value = 0.0013. The T allele of the CYP2C19*17 (rs12248560) C>T is linked to CAD with OR (95% CI) = 2.02, p-value = 0.003. In addition, the A allele of the miR-423 rs6505162 C>A SNV is linked to CAD with OR (95% CI) = 1.49, p-value = 0.036. The ELISA results indicated that the TNF-α serum levels are significantly increased in CAD patients compared to healthy controls. We conclude the TNF-α rs1800629 G>A, CYP2C19*17, and miR-423 rs6505162 C>A are potential genetic loci for CAD in the Saudi population. These findings require further verification in future studies. After being verified, our results might be utilized in genetic testing to identify individuals that are susceptible to CAD and, therefore, for whom reducing modifiable risk factors (e.g., poor diet, diabetes, obesity, and smoking) would result in prevention or delay of CAD.

A novel tetra-primer ARMS-PCR for genotyping of the OPRM1 gene rs1799971 variant associated with opioid use disorders.

OBJECTIVES: A SNV is a single nucleotide change that can occur at any point in the genome. SNVs are the most common genetic variants that occur in the human genome, and a number of SNVs have been found to be associated with human traits and disease. Researchers genotype SNVs using TaqMan technology, DNA microarray, MALDI-TOF mass spectrometry, and automated sequencing, which are expensive and time-consuming. The OPRM1 gene rs1799971 (A118G) has been identified for its association with Opioid use disorder (OUD). The present study focused on developing a single step identification test using Tetra-Primer Amplification Refractory Mutation System-PCR (T-ARMS-PCR) to detect the presence of SNV OPRM1 rs1799971 (A118G). This study was performed to optimize the protocol for the designed four primers and validate it using a total of 52 buccal samples from volunteers who are currently under rehabilitation for the drug abuse disorder. RESULTS: Utilizing 52 DNA samples, a novel T-ARMS-PCR assay was successfully developed, tested, and validated. The products of the T-ARMS PCR for rs1799971 contained 395 bp as the control band, 186 bp as G allele (variant) and 257 bp as A allele (wild type), which were observed in the gel image. The genotype frequencies for the OPRM1 gene rs1799971 (A118G) were 44% (22/52) of homozygous variant type (GG), 28.9% (15/52) of homozygous wild type (AA) and 28.9% (15/22) of heterozygous (AG). The G allele frequency was 56.7% and A allele frequency was 43.3%.